1. Field
The present application relates generally to sample collection devices, and more specifically, to collection devices suitable for low-volume extraction.
2. Related Art
Frequently, substances must be detected that are present in only trace quantities. This is true for chemical agents, such as explosives and pharmaceuticals, and biological agents, such as microorganisms or trace amounts of nucleic acids. Commonly, these agents are obtained by swabbing a surface and then the swab is used to transfer the material to a vessel containing diagnostic reagents or a solution to extract the agents of interest.
An underlying problem with this strategy is that the swab requires the agent to be removed by using a washing solution and this step dilutes the concentration of the agent making it more difficult to detect. Two strategies are generally used to alleviate this limitation:
1. If the material is solid (for example whole cells), then it is sedimented from the swab eluate by centrifugation and/or filtration prior to extraction. If the agent is soluble, then substances can be added to the washing solution to aid precipitation of the agent thereby making it amenable to concentrating by centrifugation.
2. The sample is extracted in the large volume of solution and then the extractive is concentrated for analysis.
An example where these methods can be used is in the detection of trace (i.e., low copy number (LCN)) nucleic acids in forensic samples.
There are a number of situations where strategy 1 is used. For example, in U.S. Pat. No. 6,475,165 the cells from cervical swabs are washed from the sampling device and sedimented by centrifugation. While this method is acceptable for many samples, there are instances where such a method is unacceptable. For example, if a forensic sample is degraded then it is likely that the cells are not intact and so DNA would be solublized and therefore not sedimented by centrifugation. This can be overcome by washing the collection device in ethanol or isopropanol but such an action results in the nucleic acid being presented to subsequent steps in the presence of alcohols.
The most widely used methods for trace nucleic acid sampling follow strategy 2 and commercial kits are available (DNA IQ™ (Promega, Madison, Wis., USA) and QiaAmp® (Qiagen, Valencia, Calif., USA).
One system takes the extract and then binds the nucleic acids to paramagnetic beads that are concentrated by using a magnet. Although this system is an effective method for concentrating the nucleic acid from the solvent, the procedure has many steps that can only be automated using specialist equipment. More importantly for forensic samples, it requires the tube to be opened several times and so enhances the risk of contamination from extraneous nucleic acid. Ref. 1.
The filtration/column methods use silica or charged resin columns that bind the nucleic acid. These systems also require a number of additional steps for loading, washing and elution of the nucleic acid. For automation, vacuum manifolds are often used and these present a risk of contaminating, extraneous nucleic acid material being drawn through the column. Ref. 2.
DNA extraction using these methods can be automated but in general they require customized robotic systems. For the QiaAmp® system, the Qiagen Corporation provides a robot known as the QIACube®. In the example shown in the paper by Montpetit et al., the Biorobot EZ1 performs bead-based DNA extraction/concentration. Such equipment generally can only be used for the purpose of their design and therefore can, in some cases, represent a significant outlay for a single-use device. Ref. 6.
Other methods include a concentration step using ultra-filtration through a membrane filter, for example Microcon® columns (Millipore, Bedford, Mass., USA). Ref. 3.
Yet other methods concentrate the nucleic acids by the addition of ethanol or isopropanol to precipitate the DNA or RNA. This is then separated by centrifugation.
Yet other methods concentrate the eluate by using n-butanol which in effect draws the water from the extractive.
Neither of the latter two methods can be readily automated and both risk exposing the sample to extraneous contamination.
LCN Forensic DNA Extraction
The use of LCN DNA in forensic analysis is presents several potential difficulties. Because the samples often contain little DNA (just a few genomes in some cases) the environment surrounding the tube, the people handling the samples, and the laboratory equipment are all often better sources of DNA than the sample itself. Therefore all effort must be made to protect the integrity of the sample. For example, Van Oorschot and Jones reported that DNA on laboratory equipment and on a casework samples persisted for long periods of time. Ref. 4.
A notable example of the outcome of forensic samples being compromised is the case of The Queen v. Sean Hoey, in which the defendant had been previously charged with twenty-nine counts of murder in a terrorist attack in a shopping centre in Omagh, Northern Ireland on the 15 Aug. 1988. Ref. 5. The prosecution relied on LCN DNA tests that, on re-examination, were shown to have “very many unsatisfactory matters”. These included the likelihood of cross-contamination in the laboratory with other samples and also in the recovery and storage of the items by the Army, Police and Scene of Crime Officers.
The case demonstrated the potential deficiencies in sampling, storage and laboratory handling of LCN samples, which require very exacting standards.